Project 2- Summary: Weak Th1 immune responses commonly result from therapeutic cancer vaccines, suggesting improved systems are needed to obtain robust effective clinical responses. One key feature of vaccines is the antigen, and patient-specific tumor neoantigens have led to clinical trials demonstrating that strong antigen-specific immune responses can be generated via vaccination with peptide neoantigens, although these have been consistently noted to be primarily CD4+ rather than CD8+ T cell responses. The adjuvant is also key, and CpG oligodeoxynucleotides (CpG-ODNs) and double-stranded RNA (dsRNA) have been recognized for their ability to polarize towards antitumor Th1 immune responses. A third component, and the focus of this project, is the presentation of the antigen and adjuvant. While neoantigens and CpG/dsRNA have been conventionally introduced in soluble form, preclinical data indicates co-delivery of the two is important, and that the spatial distribution of the adjuvant at the nanoscale influences TLR activation and downstream T cell polarization. Multifunctional 3D nanostructure DNA origami may provide an ideal system to controllably co- present adjuvants and antigens. We have successfully assembled DNA-origami structure square-lattice blocks (SQBs) that can control adjuvant spacing in the context of loaded antigens; our pilot data show that CpG-SQB vaccines enhance antigen cross-presentation by dendritic cells and that uniform 3.5 nm spacing of CpG on SQBs can differentially induce Th1 polarization. Here we hypothesize that co-presentation of neoantigens with adjuvants at specific valency and spacing from SQBs that induce Th1 polarization will boost the efficacy of therapeutic cancer vaccines. Our aims are: (1) Investigate how adjuvant spacing on SQBs affects Th1/Th2 immune responses in vitro. We will fabricate SQBs with short dsRNA and CpG coupled to the SQBs at defined spacings and stoichiometries, and compare how differently spaced dsRNA-SQBs with various antigen/adjuvant stoichiometries maturate DCs and induce Th1 immune responses, (2) Determine the extent to which Th1 immune responses are triggered by vaccines utilizing SQBs for adjuvant and antigen presentation in vivo. We will evaluate the capacity of CpG-SQBs and dsRNA-SQBs, when fabricated with tumor antigens, to elicit anti- tumor, Th1 immune responses in the mouse B16F10 melanoma and CT26 colon tumor models, and (3) Assess whether dsRNA-SQBs can generate robust CD8+ T cell responses against human tumor neoantigens. We will confirm that antigen delivered by dsRNA-SQBs can be presented in the context of human MHC through vaccination in the transgenic HLA-A2.1+ mouse, and test the optimized dsRNA-SQBs, loaded with human neoantigens already demonstrated to stimulate predominantly CD4+ T cell responses in patients, for ability to polarize in the direction of CD8+ T cell responses through in vitro co-culture of patient PBMCs with DNA-origami vaccine pulsed antigen-presenting cells.